The life cycle of the human immunodeficiency virus (HIV) and other retroviruses is dependent upon nuclear transport. Once in the cytoplasm, HIV RNA is reverse-transcribed into double-stranded DNA that must enter the nucleus. Other retroviral regulatory proteins also enter the nucleus to perform their function, and viral transcripts are exported to the cytoplasm. The viral regulatory proteins, Rev and Tat, have been identified as key modulators of the transcription and transport of HIV envelope mRNA out of the nucleus. We are examining the processes of nuclear protein import and export in vitro using the HIV Rev protein. The Rev protein actively shuttles between nucleolus and cytoplasm and mediates premature export of unspliced retroviral RNAs. Using digitonin permeabilized cultured cells, we developed an in vitro assay for studying both nuclear transport and nucleolar accumulation of molecules such as HIV Rev. The localization of nuclear shuttling proteins such as HIV Rev is controlled by the relative rates of nuclear import and export. We also have developed an in vitro assay for nuclear export using the green fluorescent protein-labelled Rev fused to a hormone-inducible import signal (Rev/Gr/GFP). We examined the requirements for nuclear transport and nucleolar accumulation of HIV Rev in vitro and in living cells. The data suggest that dissociation from the nucleolus is an ATP-dependent process. Utilizing Fluorescence Recovery After Photobleaching, Rev was found to be immobilized at the nucleolus, further strengthening the role for a ATP-dependent release from the nucleolus. This regulated nucleolar localization may reflect a more widespread phenomenon since it has been observed for other nucleolar proteins involved in apoptosis and cell cycle control. The system we have described should allow for the identification of the requirements for regulated nucleolar targeting.